DiaMetra Thyroglobulin Elisa Kit

PRINCIPLE
The essential reagents required for an immunoenzymometric assay include high affinity and specificity antibodies (enzyme and immobilized), with different and distinct epitope recognition, in excess, and native antigen. In this procedure, the immobilization takes place during the assay at the surface of a microplate well through the interaction of the streptavidin coated on the well and exogenously added biotinylated monoclonal Thyroglobulin antibody. When monoclonal biotinylated antibody is mixed with a serum containing the Thyroglobulin antigen and the antibody, an antibody-antigen complex is formed.

CLINICAL APPLICATIONS
Thyroglobulin (TG), a glycoprotein with a molecular weight of about 660,000 Daltons, is the thyroid’s main iodine protein and the most important compound of follicular colloid. Thyroglobulin is the form under which the active hormones T3 and T4 together with their immediate forerunners MIT and DIT are laid inside the thyroid gland. The clinical applications of the TG dosage seem to originate from its specificity for the thyroid and related cells. The dosage of TG can be used as support to scintigraphies or other techniques for studying pathogenesis, making a diagnosis and analyzing the course of thyroid disorders. The dosage of TG before and after replacement treatment with L-Thyroxin cannot be established in cases of hypothyroidism due to thyroid agenesis. In cases of secondary hypothyroidism with a dysglandular goiter or ectopic thyroid, the levels of TG are normal or high. The circulating levels of TG tend to increase in several thyroid disorders such as toxic and atoxic goiter, subacute thyroiditis, Basedow’s disease and carcinoma. In Basedow’s disease the TG dosage is a potentially interesting index of normalization of hyperthyroidism in patients treated with anti-thyroid drugs. In the oncology field and more specifically for differentiated thyroid carcinoma, there are very promising applications linked to the ability of thyroid tumors tissues to concentrate iodine and synthesize TG as a normal thyroid. Basically the dosage of TG can be used as follows: 
a. Pre-operating diagnosis of thyroid tumors. This application does not allow the differentiated diagnosis of the tumor as the values of TG seen in malignant and benign nodules are superimposable.
b. Post-operation monitoring In patients treated surgically or with radiotherapy, long lasting TG levels suggest the presence of a residual carcinoma and/or carcinoma with metastasis.
c. Monitoring of totally thyroidectomized patients The use of circulating TG as an indicator of recurrent tumors (metastasis marker) has an established clinical value: the increase of Thyroglobulinaemia indicates the need to undergo further analysis for confirming the diagnosis. Interesting advantages can come from: a) a reduced use of scintigraphic diagnostic techniques as they imply regular suspension of replacement treatment and frequent exposure to radiation, b) and complete completion of the information obtained via scintigraphy.

PRECAUTIONS 
• Please adhere strictly to the sequence of pipetting steps provided in this protocol. The performance data represented here were obtained using specific reagents listed in this Instruction For Use. 
• All reagents should be stored refrigerated at 2- 8°C in their original container. Any exceptions are clearly indicated. The reagents are stable until the expiry date when stored and handled as indicated. 
• Allow all kit components and specimens to reach room temperature (22-28°C) and mix well prior to use. 
• Do not interchange kit components from different lots. The expiry date printed on box and vials labels must be observed. Do not use any kit component beyond their expiry date. 
• If you use automated equipment, the user has the responsibility to make sure that the kit has been appropriately tested. 
• The incomplete or inaccurate liquid removal from the wells could influence the assay precision and/or increase the background. To improve the performance of the kit on automatic systems, it is recommended to increase the number of washes. 
• It is important that the time of reaction in each well is held constant for reproducible results. Pipetting of samples should not extend beyond ten minutes to avoid assay drift. If more than 10 minutes are needed, follow the same order of dispensation. If more than one plate is used, it is recommended to repeat the dose response curve in each plate.
• Addition of the TMB Substrate solution initiates a kinetic reaction, which is terminated by the addition of the Stop Solution. Therefore, the TMB Substrate and the Stop Solution should be added in the same sequence to eliminate any time deviation during the reaction.
• Observe the guidelines for performing quality control in medical laboratories by assaying controls and/or pooled sera. • Maximum precision is required for reconstitution and dispensation of the reagents.
• Samples microbiologically contaminated, highly lipemeic or haemolysed should not be used in the assay. 
• Plate readers measure vertically. Do not touch the bottom of the wells.

QUALITY CONTROL
Each laboratory should assay controls at normal, high and low levels range of hypothyroid, euthyroid and hyperthyroid for monitoring assay performance. These controls should be treated as unknowns and values determined in every test procedure performed. Quality control charts should be maintained to follow the performance of the supplied reagents. Pertinent statistical methods should be employed to ascertain trends. The individual laboratory should set acceptable assay performance limits. Other parameters that should be monitored include the 80, 50 and 20% intercepts of the calibration curve for run-to-run reproducibility. In addition, maximum absorbance should be consistent with past experience. Significant deviation from established performance can indicate unnoticed change in experimental conditions or degradation of kit reagents. Fresh reagents should be used to determine the reason for the variations.

CALCULATION OF RESULTS
A dose response curve is used to ascertain the concentration of human thyroglobulin (Tg) in unknown specimens.
1. Record the absorbance obtained from the printout of the microplate reader.
2. Plot the absorbance for each duplicate serum reference versus the corresponding Tg concentration in ng/mL on linear graph paper (do not average the duplicates of the serum references before plotting)
3. Draw the best fit curve through the plotted points.
4. To determine the concentration of Tg for an unknown locate the average absorbance of the duplicates for each unknown on the vertical axis of the graph, find the intersecting point on the curve, and read the concentration (in ng/mL) from the horizontal axis of the graph (the duplicates of the unknown may be averaged as indicated). In the following example, the average absorbance (0.424) intersects the dose response curve at 25.2 ng/mL Tg concentration.
Note: computer data reduction software designed for ELISA assays may also be used for the data reduction. If such software is utilized, the validation of the software should be ascertained.